N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Toxicity to terrestrial arthropods

Administrative data

Endpoint:

toxicity to bees: acute contact

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994/07/19 to 1994/07/29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed under GLP conditions. The guidelines followed in this study are EPPO Guideline No 170 (1992). No deviations occurred from these guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPPO Guideline No 170

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Application method:

other: Oral and contact

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

EC Number:

266-257-8

EC Name:

N-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Cas Number:

66215-27-8

Molecular formula:

C6H10N6

IUPAC Name:

N2-cyclopropyl-1,3,5-triazine-2,4,6-triamine

Specific details on test material used for the study:

The test material is cyromazine.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Young workerbees of the honeybee (Apis mellifera) were treated with varied concentrations (concentration series) of the test substance and of the reference substance. The treated bees were kept under controlled climatic conditions and assessed for toxic effects up to 48 hours. Two different treatments were used permitting the evaluation of toxic feeding effects and contact effects of the test substance and the reference substance.

For the oral toxicity test, the bees were fed with a defined quantity of a 50 % aqueous sucrose solution that contained varied concentrations (concentration series) of the test substance. The test was performed by way of group feeding: ca. 0.2 mL (= 0.251 g) of the test solution were offered to ten bees per cage. After feeding the food tube was reweighed to control the eaten up quantity of the test solution.

For the contact toxicity test, the compounds were dissolved in acetone. With anaesthetized bees were treated individually by topical application with an Eppendorf Micropipette. 10 of the test solution resp. 1 of the reference solution were dosed to the thorax of each bee. After application, the test cages were supplied with water and food tubes (with candy) and transferred to the climatic room.

Test substrate

Vehicle:

yes

Remarks:

Methanol for the contact toxicity test (acetone was used for the reference toxic item dimethoate in the contact toxicity test)

Details on preparation and application of test substrate:

The exactly measured amounts of the substances were mixed with sucrose solution or methanol substance) immediately before application.

Test organisms

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ANIMALS
- Source: External Non-Lab source
- Age at study initiation: adult worker bees
- Diet: Sugar/honey solution available throughout 48 hour test duration
- Acclimation period: Acclimatized to the test conditions for 1-2 hours before treatment

Study design

Study type:

laboratory study

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

26 - 28°C

Humidity:

47 - 48% as relative humidity

Photoperiod and lighting:

8 hours diffuse light daily

Details on test conditions:

The test chambers consisted of small-ventilated boxes with a frontal glass plate for observation. Ten bees were placed into each chamber and three chambers were established for each treatment. Food (a sugar/honey solution) was available throughout the 48 hours duration of the test and the bees were monitored for toxic effects after 24 and 48 hours.

The test bees were anaesthetized with CO2 and treated, via topical application to the thorax with 10µL of the test solution or 1µL of the Dimethoate being applied to each bee. After application the bees were allowed free access to food and water for the remaining duration of the test. Cyromazine (2.5, 5.0, 10, 15, 20 mg/mL) was dissolved in methanol before application and the positive dimethoate controls (62.5, 125, 250, 500 µg/mL) were dissolved in acetone before application. Further negative control groups were treated with 1 µL acetone per bee or 10 µL of methanol per bee.

Test cages were disposable cages of cardboard with holes in the bottom for ventilation and a glass plate in front for observation of the bees (dimensions, inside: 80 mm x 45 mm x 65 mm), 10 honeybees per cage, 3 cages per concentration, 30 honeybees per concentration, bees were fed continuously during the test candy (mixture of sugar and honey) and water.

Nominal and measured concentrations:

Oral toxicity test
Nominal concentrations (µg/bee): 80, 120, 160, 200, 240, 280
Dimethoate positive controls (µg/bee): 0.2, 0.24, 0.28, 0.32, 0.36, 0.40

Contact toxicity test
Nominal concentrations (µg/bee): 25, 50, 100, 150, 200
Dimethoate positive controls (µg/bee): 0.0625, 0125, 0.25, 0.5, 1.0

Reference substance (positive control):

yes

Remarks:

Dimethoate

Results and discussion

Toxic reference:

Dimethoate

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

186 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Oral toxicity test

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 200 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Contact toxicity test

Details on results:

No behavioural anomalies were observed

Physical parameters monitored: The test temperature was 26-28°C with 47-48% R H and 8 hours diffuse light daily.

Results with reference substance (positive control):

The LD50 for the contact toxicity test of the reference substance Dimethoate product after 24 hours was 0.26 µg/bee, and for 48 hours are 0.25 µg/bee. For the oral toxicity test, the 24 hour LD50 was 0.35 µg/bee and the 48 hour was 0.34 µg/bee.

Reported statistics and error estimates:

The determination of the medial lethal dose value (LD50) was carried out by non-linear regression analysis using a special computer programme.

Any other information on results incl. tables

Table 1

Acute contact toxicity of test material on the adult honeybee (Apis Mellifera)

Treatment	Concentration of the test  solution (mg/mL)	Dose (µg/bee)	% Mortality after
			24h	48 h
Control			0	0
Test material	2 5	25	13 3	13 3
	5 0	50	13 3	16 7
	10	100	6 7	13 3
	15	150	3 3	3 3
	20	200	6 7	16 7
Control			3 3	3 3
Dimethoate	0 625	0 0625	0	0
	0 125	0 125	0	0
	0 250	0 25	46 7	53 3
	0 500	0 5	96 7	96 7
		1 0	100 0	100 0

 

Table 2

Acute oral toxicity of cyromazine on the adult honeybee (Apis Mellifera)

 

Treatment	Concentration of the test solution (mg/mL)	Dose (µg/bee)	% Mortality after
			24h	48 h
Sucrose solution			0	0
Cyromazine	4	80	0	0
	6	120	10 0	10 0
	8	160	23 3	23 3
	10	200	56 7	60 0
	12	240	86 7	86 7
	14	280	90 0	90 0
Dimethoate	0 01	0 20	6 7	6 7
	0 012	0 24	0	3 3
	0 014	0 28	23 3	30 0
	0 016	0 32	36 7	36 7
	0 018	0 36	50 0	56 7
	0 020	0 40	73 3	80 0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour LD50 of cyromazine with adult honeybees was determined to be >200 µg/bee when applied topically.

Executive summary:

Two tests, each with a duration of 48 hours, were conducted to determine the LD₅₀ of the test substance to the honeybee (Apis mellifera) in the oral toxicity test and in the contact toxicrty test. The study was performed according to EPPO Guideline No. 170. Test criteria were mortality and the behaviour of the honeybees in comparison with the control. In the contact toxicity test cyromazine showed only negligible effect on honeybee mortality at dosages up to 200 μg/bee. 

 

The (48-h) in the contact toxicity test was greater than 200 μg/bee the highest concentration tested. The LD₅₀ of the test substance cyromazine in the oral toxicity test was 187μg substance per bee after 24 hours and 186 μg substance per bee after 48 hours. No behavioural anomalies were observed. The LD₅₀ of the reference substance Dimethoate product after 24 hours was 0.26 μg substance per bee in the oral toxicity test and 0.16 μg substance per bee in the contact toxicity test.